Soybean event MON89788 and methods for detection thereof

ABSTRACT

The present invention provides for soybean plant and seed comprising transformation event MON89788 and DNA molecules unique to these events. The invention also provides methods for detecting the presence of these DNA molecules in a sample.

This application is a continuation of U.S. application Ser. No.13/214,081, filed Aug. 19, 2011 (pending), which application is adivisional of U.S. application Ser. No. 12/575,352, filed Oct. 7, 2009,now U.S. Pat. No. 8,053,184, which application is a divisional of U.S.application Ser. No. 11/441,914, filed May 26, 2006, now U.S. Pat. No.7,632,985, which application claims the benefit of U.S. ProvisionalApplication No. 60/685,584, filed May 27, 2005, each of the entiredisclosures of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a new and distinctive transgenicsoybean transformation event, designated MON89788, a soybean cultivarderived therefrom, and plant parts, seed, and products thereof. Theinvention also relates to assays for detecting the presence of a DNAmolecule specific to MON89788 in a plant part extract or seed extract.

2. Description of Related Art

Soybean (Glycine max) is an important crop in many areas of the world.The methods of biotechnology have been applied to soybean forimprovement of the agronomic traits and the quality of the product. Onesuch agronomic trait important in soybean production is herbicidetolerance, in particular, tolerance to glyphosate herbicide. A herbicidetolerant soybean event would be a useful trait for managing weeds.

N-phosphonomethylglycine, also known as glyphosate, is a well-knownherbicide that has activity on a broad spectrum of plant species.Glyphosate is the active ingredient of Roundup® (Monsanto Co., St.Louis, Mo.), a safe herbicide having a desirably short half-life in theenvironment. When applied to a plant surface, glyphosate movessystemically through the plant. Glyphosate is phytotoxic due to itsinhibition of the shikimic acid pathway, which provides a precursor forthe synthesis of aromatic amino acids. Glyphosate inhibits the enzyme5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) found in plants.

Glyphosate tolerance can be achieved by the expression of EPSPS variantsthat have lower affinity for glyphosate and therefore retain theircatalytic activity in the presence of glyphosate (U.S. Pat. Nos.5,633,435; 5,094,945; 4,535,060, and 6,040,497). Enzymes that degradeglyphosate in plant tissues (U.S. Pat. No. 5,463,175) are also capableof conferring cellular tolerance to glyphosate. Such genes are used forthe production of transgenic crops that are tolerant to glyphosate,thereby allowing glyphosate to be used for effective weed control withminimal concern of crop damage. For example, glyphosate tolerance hasbeen genetically engineered into corn (U.S. Pat. No. 5,554,798), wheat(U.S. Pat. No. 6,689,880), cotton (U.S. Pat. No. 6,740,488), soybean (WO9200377) and canola (US Patent Appl. 20040018518). The transgenes forglyphosate tolerance and the transgenes for tolerance to otherherbicides, e.g. the bar gene, (Toki el al., 1992; Thompson et al.,1987; phosphinothricin acetyltransferase (DeBlock et al., 1987), fortolerance to glufosinate herbicide) are also useful as selectablemarkers or scorable markers and can provide a useful phenotype forselection of plants linked with other agronomically useful traits.

The expression of foreign genes in plants is known to be influenced bytheir chromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., 1988).For this reason, it is often necessary to screen a large number ofevents in order to identify an event characterized by optimal expressionof an introduced gene of interest. For example, it has been observed inplants and in other organisms that there may be a wide variation inlevels of expression of an introduced gene among events. There may alsobe differences in spatial or temporal patterns of expression, forexample, differences in the relative expression of a transgene invarious plant tissues, that may not correspond to the patterns expectedfrom transcriptional regulatory elements present in the introduced geneconstruct. For this reason, it is common to produce hundreds tothousands of different events and screen those events for a single eventthat has desired transgene expression levels and patterns for commercialpurposes. An event that has desired levels or patterns of transgeneexpression is useful for introgressing the transgene into other geneticbackgrounds by sexual outcrossing using conventional breeding methods.Progeny of such crosses maintain the transgene expressioncharacteristics of the original transformant. This strategy is used toensure reliable gene expression in a number of varieties that are welladapted to local growing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the pre-market approval and labeling of foods derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well known polynucleic acid detectionmethod such as the polymerase chain reaction (PCR) or DNA hybridizationusing polynucleic acid probes. These detection methods generally focuson frequently used genetic elements, such as promoters, terminators,marker genes, etc. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNA(“flanking DNA”) adjacent to the inserted transgene DNA is known. Anevent-specific PCR assay is discussed, for example, by Windels et al.(1999), who identified glyphosate tolerant soybean event 40-3-2 by PCRusing a primer set spanning the junction between the insert transgeneand flanking DNA, specifically one primer that included sequence fromthe insert and a second primer that included sequence from flanking DNA.Transgenic plant event specific DNA detection methods have also beendescribed in U.S. Pat. Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974and 6,689,880; 6,900,014 and 6,818,807, herein incorporated by referencein their entirety.

This invention relates to the glyphosate tolerant soybean event MON89788(also referred to as MON19788 or GM_A19788) and to the DNA moleculescontained in these soybean plants that are useful in detection methodsfor the plant and progeny thereof and plant tissues derived fromMON89788.

SUMMARY OF THE INVENTION

The present invention provides a soybean transgenic event designatedMON89788 (also referred to as MON19788) and progeny thereof havingrepresentative seed deposited with American Type Culture Collection(ATCC) with accession No. PTA-6708. Another aspect of the invention isthe plant cells or regenerable parts of the plant and seeds of thesoybean event MON89788. The invention also includes plant parts ofsoybean event MON89788 that include, but are not limited to a cell,pollen, ovule, flowers, shoots, roots, leaves, and products derived fromMON89788, for example soybean meal, flour and oil.

One aspect of the invention provides compositions and methods fordetecting the presence of a DNA transgene/genomic junction region from asoybean event MON89788 plant or seed or products derived from plantparts or seed. DNA molecules are provided that comprise at least onetransgene/genomic junction DNA molecule selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2, and complements thereof,wherein the junction molecule spans the insertion site that comprises aheterologous DNA inserted into the genome of the soybean cell and thegenomic DNA from the soybean cell flanking the insertion site soybeanevent MON89788. Such junction sequences may, in one aspect of theinvention, be defined as comprising nucleotides 1093-1113 or 5396-5416of SEQ ID NO:9, respectively. In other aspects of the invention, thejunctions may be defined as including additional portions of theflanking genome and transgene, for example, and may be defined ascomprising one or more sequence as given by nucleotides 1073-1113,1043-1113, 1093-1133, 1093-1163, 1043-1163, 5376-5416, 5346-5416,5396-5436, 5396-5416, 5396-5466, or 5346-5466 of SEQ ID NO:9. Suchsequences and plants and seeds comprising these sequences therefore formone aspect of the invention.

A novel DNA molecule is provided that is a DNA transgene/genomic regionSEQ ID NO:3 or the complement thereof, from soybean event MON89788. Asoybean plant and seed comprising SEQ ID NO:3 in its genome is an aspectof this invention. SEQ ID NO:3 further comprises SEQ ID NO:1 in itsentirety.

According to another aspect of the invention, a DNA molecule is providedthat is a DNA transgene/genomic region SEQ ID NO:4, or the complementthereof, wherein this DNA molecule is novel in soybean event MON89788. Asoybean plant and seed comprising SEQ ID NO:4 in its genome is an aspectof this invention. SEQ ID NO:4 further comprises SEQ ID NO:2 in itsentirety.

According to another aspect of the invention, two nucleic acid moleculesare provided for use in a DNA detection method, wherein the firstnucleic acid molecule comprises at least 11 or more contiguouspolynucleotides of any portion of the transgene region of the DNAmolecule of SEQ ID NO:3 and the second nucleic acid is a molecule ofsimilar length of any portion of a 5′ flanking soybean genomic DNAregion of SEQ ID NO:3, wherein these nucleic acid molecules when usedtogether are useful as primers in a DNA amplification method thatproduces an amplicon. The amplicon produced using these primers in theDNA amplification method is diagnostic for soybean event MON89788 DNA.The amplicon produced by the described primers that is homologous orcomplementary to a portion of SEQ ID NO:3 comprising SEQ ID NO:1 is anaspect of the invention.

According to another aspect of the invention, two nucleic acid moleculesare provided for use in a DNA detection method, wherein the firstnucleic acid molecule comprises at least 11 or more contiguouspolynucleotides of any portion of the transgene region of the DNAmolecule of SEQ ID NO:4 and a second nucleic acid molecule of similarlength of any portion of a 3′ flanking soybean genomic DNA of SEQ IDNO:4, wherein these nucleic acid molecules when used together are usefulas primers in a DNA amplification method that produces an amplicon. Theamplicon produced using these primers in the DNA amplification method isdiagnostic for soybean event MON89788 DNA. The amplicon produced by thedescribed primers that is homologous or complementary to a portion ofSEQ ID NO:4 comprising SEQ ID NO:2 is an aspect of the invention.

Any nucleic acid primer pair derived from SEQ ID NO:3 or SEQ ID NO:4, orSEQ ID NO:9 or the complements thereof, that when used in a DNAamplification reaction produces an amplicon diagnostic for soybean eventMON89788-derived tissue, such as an amplicon that comprises SEQ ID NO:1or SEQ ID NO:2 or any portion of SEQ ID NO:9 respectively, is anotherembodiment of the invention. In a particular embodiment, the primer pairmay consist of primer A (SEQ ID NO:5) and primer D (SEQ ID NO:8).

Another aspect of the invention is a soybean plant, or seed, or productderived from a plant or seed comprising event MON89788, in which thegenomic DNA when isolated from the soybean plant, or seed, or productproduces an amplicon in a DNA amplification method that comprises SEQ IDNO:1 or SEQ ID NO:2.

Still another aspect of the invention is a soybean plant, or seed, orproduct derived from a plant or seed comprising MON89788, in which thegenomic DNA when isolated from the soybean plant, or seed, or productproduces an amplicon in a DNA amplification method, wherein DNA primermolecules SEQ ID NO:5 and SEQ ID NO:6 are used in the DNA amplificationmethod.

Yet another aspect of the invention is a soybean plant, seed, product,or commodity derived from the plant or seed, comprising MON89788, inwhich the genomic DNA when isolated from the soybean plant, or seed, orproduct produces an amplicon in a DNA amplification method, wherein DNAprimer molecules SEQ ID NO:7 and SEQ ID NO:8 are used in the DNAamplification method. The product or commodity may comprise, withoutlimitation, a food or feed product comprising or derived from one ormore of the following products of a soybean plant comprising eventMON89788: lecithin, fatty acids, glycerol, sterol, edible oil, defattedsoy flakes, soy meals including defatted and toasted soy meals, soy milkcurd, tofu, soy flour, soy protein concentrate, isolated soy protein,hydrolyzed vegetable protein, textured soy protein, and soy proteinfiber.

According to another aspect of the invention, a method of detecting thepresence of DNA corresponding specifically to the soybean event MON89788DNA in a sample is provided. Such method comprising: (a) contacting asample comprising DNA with a DNA primer pair; (b) performing a nucleicacid amplification reaction, thereby producing the amplicon; and (c)detecting the amplicon, wherein said amplicon comprises SEQ ID NO:1 orSEQ ID NO:2. A kit comprising DNA primer molecules that when used in aDNA amplification method produces an amplicon comprising SEQ ID NO:1 orSEQ ID NO:2 is a further aspect of the invention.

According to another aspect of the invention, a method of detecting thepresence of DNA corresponding specifically to the soybean event MON89788DNA in a sample is provided. Such method comprising: (a) contacting asample comprising DNA with a probe that hybridizes under stringenthybridization conditions with genomic DNA from soybean event MON89788and does not hybridize under the stringent hybridization conditions witha control soybean plant DNA; (b) subjecting the sample and probe tostringent hybridization conditions; and (c) detecting hybridization ofthe probe to the soybean event MON89788 DNA, wherein said probecomprises SEQ ID NO:1 or SEQ ID NO:2. The sample may comprise a progenyseed, plant, or plant part comprising soybean event MON89788, or any ofthe following products derived from a plant comprising MON89788:lecithin, fatty acids, glycerol, sterol, edible oil, defatted soyflakes, soy meals including defatted and toasted soy meals, soy milkcurd, tofu, soy flour, soy protein concentrate, isolated soy protein,hydrolyzed vegetable protein, textured soy protein, and soy proteinfiber. A kit comprising a DNA probe comprising a DNA molecule that ishomologous or complementary to SEQ ID NO:1 or SEQ ID NO:2 is an aspectof the invention. A kit comprising a DNA molecule comprising SEQ IDNO:18, SEQ ID NO:19, or SEQ ID NO:20, or their complements, is also anaspect of the invention.

According to another aspect of the invention, a method of producing asoybean plant that tolerates an application of glyphosate is providedthat comprise the steps of: (a) sexually crossing a first parentalglyphosate tolerant soybean plant comprising event MON89788, and asecond parental soybean plant that lacks the glyphosate tolerance,thereby producing a plurality of progeny plants; and (b) selecting aprogeny plant that tolerates application of glyphosate. Breeding methodsmay additionally comprise the steps of crossing the parental plantcomprising soybean event MON89788 to a second parental soybean plantthat is also tolerant to glyphosate and selecting for glyphosatetolerant progeny by molecular marker DNA genetically linked to theglyphosate tolerant phenotype found in each parent.

Another aspect of the invention is a method to control weeds in a fieldof soybean plants comprising MON89788, wherein said method comprisesplanting a field with soybean seed comprising event MON89788 saidrepresentative seed deposited as ATCC accession No. PTA-6708, allowingsaid seed to germinate and treating said plants with an effective doseof glyphosate to control weed growth in said field.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Organization of the transgene insertion in the genome of asoybean plant comprising event MON89788.

FIG. 2A-2B. Processing of commodity products from soybean.

DETAILED DESCRIPTION

The present invention relates to a novel soybean transformation eventdesignated MON89788 that provides glyphosate tolerance, and the plantparts and seed and products produced from plants, plant parts, seed, andproducts comprising the event. The invention provides DNA molecules thatare novel in the genome of soybean cells comprising MON89788 and DNAmolecules that can be used in various DNA detection methods to identifyMON89788 DNA in a sample. The invention provides a method to controlweeds in a field of plants containing MON89788 by treating the weeds inthe field comprising plants comprising event MON89788 with a glyphosateherbicide.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al. (1991) and Lewin (1994). Thenomenclature for DNA bases as set forth at 37 CFR §1.822 is used.

As used herein, the term “soybean” means Glycine max and includes allplant varieties that can be bred with soybean.

As used herein, the term “comprising” means “including but not limitedto”.

“Glyphosate” refers to N-phosphonomethylglycine and its salts,Glyphosate is the active ingredient of Roundup® herbicide (MonsantoCo.). Treatments with “glyphosate herbicide” refer to treatments withthe Roundup®, Roundup Ultra®, Roundup Pro® herbicide or any otherherbicide formulation containing glyphosate. Examples of commercialformulations of glyphosate include, without restriction, those sold byMonsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX,ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE,RODEO®, POLARIS®, SPARK® and ACCORD® herbicides, all of which containglyphosate as its isopropylammonium salt; ROUNDUP® WEATHERMAX(glyphosate potassium salt), those sold by Monsanto Company as ROUNDUP®DRY and RIVAL® herbicides, which contain glyphosate as its ammoniumsalt; that sold by Monsanto Company as ROUNDUP® GEOFORCE, which containsglyphosate as its sodium salt; and that sold by Syngenta Crop Protectionas TOUCHDOWN® herbicide, which contains glyphosate as itstrimethylsulfonium salt. Treatment of a field comprising glyphosatetolerant soybean plants comprising event MON89788 with any of theseglyphosate herbicide formulations will control weed growth in the fieldand not affect the growth or yield of the soybean plants comprisingMON89788.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, for example, a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatinclude the heterologous transgene DNA and the flanking genomic DNA. Theterm “event” also refers to DNA from the original transformantcomprising the inserted DNA and flanking genomic sequence immediatelyadjacent to the inserted DNA that would be expected to be transferred toa progeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (for example, the original transformant and progenyresulting from the selling) and a parental line that does not containthe inserted DNA.

A glyphosate tolerant soybean plant can be bred by first sexuallycrossing a first parental soybean plant consisting of a soybean plantgrown from a transgenic glyphosate tolerant soybean plant comprisingMON89788 or an soybean plant that is a progeny of the cross of such aplant that expresses the glyphosate tolerant phenotype, and a secondparental soybean plant that lacks the tolerance to glyphosate, therebyproducing a plurality of first progeny plants; and then selecting aprogeny plant that is tolerant to application of glyphosate herbicide.These steps can further include the back-crossing of the glyphosatetolerant progeny plant to the second parental soybean plant or a thirdparental soybean plant, then selecting progeny by application withglyphosate or by identification with molecular markers associated withthe trait thereby producing an soybean plant that tolerates theapplication of glyphosate herbicide. Molecular markers may be used thatcomprise the junction DNA molecules identified at the 5′ and 3′ sites ofinsertion of the transgene in event MON89788.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating, exogenous transgenes. Back-crossing to a parental plant andout-crossing with a non-transgenic plant as previously described is alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in one of several references, e.g., Fehr, (1987).

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, for example, aradioactive isotope, a ligand, a chemiluminescent agent, or an enzyme.Such a probe is complementary to a strand of a target nucleic acid, inthe case of the present invention, to a strand of genomic DNA from asoybean plant comprising event MON89788 whether from a soybean plant orseed or from a sample or extract of the plant or seed that includes DNAfrom the event. Probes according to the present invention include notonly deoxyribonucleic or ribonucleic acids, but also polyamides andother probe materials that bind specifically to a target DNA sequenceand can be used to detect the presence of that target DNA sequence.

“Primers” are isolated polynucleic acids that are annealed to acomplementary target polynucleic acid strand by nucleic acidhybridization to form a hybrid between the primer and the targetpolynucleic acid strand, then extended along the target polynucleic acidstrand by a polymerase, for example, a DNA polymerase. Primer pairs ofthe present invention refer to their use for amplification of a targetpolynucleic acid molecule, for example, by the polymerase chain reaction(PCR) or other conventional nucleic acid amplification methods.

Probes and primers are generally 11 polynucleotides or more in length,preferably 18 polynucleotides or more, more preferably 24polynucleotides or 30 polynucleotides or more. Such probes and primershybridize specifically to a target molecule under high stringencyhybridization conditions. Preferably, probes and primers according tothe present invention have complete sequence identity with the targetmolecule, although probes differing from the target sequence and thatretain the ability to hybridize to target sequences under highstringency conditions may be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (1989); Ausubel et al. (1992); and Innis etal. (1990). PCR-primer pairs (a primer set) can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer (Version 0.5, ©1991, Whitehead Institute forBiomedical Research, Cambridge, Mass.).

Primers and probes based on the flanking genomic DNA and insertsequences disclosed herein (SEQ ID NOs:1-4 and 9) can be used to confirmand, if necessary, to correct the disclosed sequences by conventionalmethods, for example, by isolating the corresponding DNA molecule from adeposit of seed comprising MON89788, and determining the nucleic acidsequence such molecules. Additional associated DNA molecules may beisolated from the genome of a cell comprising MON89788 that comprise thetransgene insert and genomic flanking regions, and fragments of thesemolecules may be used as primers or probes.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from the MON89788 event in a sample.Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure andare of sufficient length to maintain this structure under highstringency conditions. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if they exhibit completecomplementarity. As used herein, molecules are said to exhibit “completecomplementarity” when every nucleotide of one of the molecules iscomplementary to a nucleotide of the other. Two molecules are said to be“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are said to be “complementary” if they can hybridize to oneanother with sufficient stability to permit them to remain annealed toone another under conventional “high-stringency” conditions.Conventional stringency conditions are described by Sambrook et al.,1989, and by Haymes et al. (1985). Departures from completecomplementarity are therefore permissible, as long as such departures donot completely preclude the capacity of the molecules to form adouble-stranded structure. In order for a nucleic acid molecule to serveas a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NOs:1-4, and 9 complements thereof or fragments of either undermoderately stringent conditions, for example at about 2.0×SSC and about65° C. In a particularly preferred embodiment, a nucleic acid of thepresent invention will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NOs:1-4, and 9 complementsthereof or fragments of either under high stringency conditions. In oneaspect of the present invention, a preferred marker nucleic acidmolecule of the present invention comprises the nucleic acid sequence asset forth in SEQ ID NO:1 or SEQ ID NO:2 or complements thereof orfragments of either. In another aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 80% and 100% or 90% and 100% sequence identity with the nucleicacid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or complementsthereof or fragments of either. Molecular marker DNA molecules thatcomprise SEQ ID NO:1, or SEQ ID NO:2, or complements thereof orfragments of either may be used as markers in plant breeding methods toidentify the progeny of genetic crosses similar to the methods describedfor simple sequence repeat DNA marker analysis, in Cregan et al. (1997);all of which is herein incorporated by reference in its' entirely. Thehybridization of the probe to the target DNA molecule can be detected byany number of methods known to those skilled in the art, these caninclude, but are not limited to, fluorescent tags, radioactive tags,antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (forexample, by PCR) using a particular amplification primer pair,“stringent conditions” are conditions that permit the primer pair tohybridize only to the target nucleic-acid sequence to which a primerhaving the corresponding wild-type sequence (or its complement) wouldbind and preferably to produce a unique amplification product, theamplicon, in a DNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thesoybean plant resulting from a sexual cross contains transgenic eventMON89788 or whether a soybean sample collected from a field comprisesMON89788, or a soybean extract, such as a meal, flour or oil comprisesMON89788. DNA extracted from a soybean plant tissue sample or extractmay be subjected to a nucleic acid amplification method using a primerpair that includes a primer derived from the genomic region adjacent tothe insertion site of inserted heterologous transgene DNA, and a secondprimer derived from the inserted heterologous transgene DNA to producean amplicon that is diagnostic for the presence of the event DNA. Theamplicon is of a length and has a sequence that is also diagnostic forthe event. The amplicon may range in length from the combined length ofthe primer pairs plus one nucleotide base pair, or plus about fiftynucleotide base pairs, or plus about two hundred-fifty nucleotide basepairs, or plus about three hundred-fifty nucleotide base pairs or more.

Alternatively, a primer pair can be derived from flanking genomicsequence on both sides of the inserted DNA so as to produce an ampliconthat includes the entire insert nucleotide sequence. A member of aprimer pair derived from the plant genomic sequence may be located adistance from the inserted transgene DNA molecule, this distance canrange from one nucleotide base pair up to about twenty thousandnucleotide base pairs. The use of the term “amplicon” specificallyexcludes primer dimers that may be formed in the DNA thermalamplification reaction.

Nucleic acid amplification can be accomplished by any of the variousnucleic acid amplification reaction methods known in the art, includingthe polymerase chain reaction (PCR). A variety of amplification methodsare known in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in Innis et al. (1990). PCR amplificationmethods have been developed to amplify up to 22 kb of genomic DNA and upto 42 kb of bacteriophage DNA (Cheng et al., 1994). These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the present invention. The sequence of theheterologous DNA insert or flanking sequence from soybean event MON89788and can be verified, and corrected if necessary by amplifying suchmolecules from the event genome using primers derived from the sequencesprovided herein followed by standard DNA sequencing methods applied tothe PCR amplicon or to isolated cloned transgene/genomic DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.,1994) where an DNA oligonucleotide is designed which overlaps both theadjacent flanking genomic DNA sequence and the inserted DNA transgenesequence. The oligonucleotide is immobilized in wells of a microwellplate. Following PCR of the region of interest (using one primer in theinserted sequence and one in the adjacent flanking genomic sequence), asingle-stranded PCR product can be hybridized to the immobilizedoligonucleotide and serve as a template for a single base extensionreaction using a DNA polymerase and labelled ddNTPs specific for theexpected next base. Readout may be fluorescent or ELISA-based. A signalindicates presence of the insert/flanking genomic sequence due tosuccessful amplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(2000). In this method an oligonucleotide is designed that overlaps theadjacent genomic DNA and insert DNA junction. The oligonucleotide ishybridized to single-stranded PCR product from the region of interest(one primer in the inserted sequence and one in the flanking genomicsequence) and incubated in the presence of a DNA polymerase, ATP,sulfurylase, luciferase, apyrase, adenosine 5′ phosphosulfate andluciferin. DNTPs are added individually and the incorporation results ina light signal which is measured. A light signal indicates the presenceof the transgene insert/flanking sequence due to successfulamplification, hybridization, and single or multi-base extension.

Fluorescence Polarization as described by Chen et al. (1999) is a methodthat can be used to detect the amplicon of the present invention. Usingthis method an oligonucleotide is designed which overlaps the genomicflanking and inserted DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted DNA and one in the flanking genomic DNA sequence) andincubated in the presence of a DNA polymerase and a fluorescent-labeledddNTP. Single base extension results in incorporation of the ddNTP.Incorporation can be measured as a change in polarization using afluorometer. A change in polarization indicates the presence of thetransgene insert/flanking genomic sequence due to successfulamplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of the flankinggenomic/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi et al. (1996) Briefly, a FRET oligonucleotide probeis designed that overlaps the flanking genomic and insert DNA junction.The unique structure of the FRET probe results in it containingsecondary structure that keeps the fluorescent and quenching moieties inclose proximity. The FRET probe and PCR primers (one primer in theinsert DNA sequence and one in the flanking genomic sequence) are cycledin the presence of a thermostable polymerase and dNTPs. Followingsuccessful PCR amplification, hybridization of the FRET probe to thetarget sequence results in the removal of the probe secondary structureand spatial separation of the fluorescent and quenching moieties. Afluorescent signal results. A fluorescent signal indicates the presenceof the flanking genomic/transgene insert sequence due to successfulamplification and hybridization.

Other described methods, such as, microfluidics (US Patent Pub.2006068398, U.S. Pat. No. 6,544,734) provide methods and devices toseparate and amplify DNA samples. Optical dyes used to detect andquantitate specific DNA molecules (WO/05017181). Nanotube devices(WO/06024023) that comprise an electronic sensor for the detection ofDNA molecules or nanobeads that bind specific DNA molecules and can thenbe detected are useful for detecting DNA molecules of the presentinvention.

DNA detection kits can be developed using the compositions disclosedherein and the methods described or known in the art of DNA detection.The kits are useful for the identification of soybean event DNA in asample and can be applied to methods for breeding soybean plantscontaining DNA. The kits may contain DNA primers or probes that arehomologous or complementary to SEQ ID NOs:1-4 and 9 or DNA primers orprobes homologous or complementary to DNA contained in the transgenegenetic elements of DNA, these DNA sequences can be used in DNAamplification reactions or as probes in a DNA hybridization method. Thestructure of the DNA of the transgene genetic elements contained in thesoybean genome and illustrated in FIG. 1 comprises a 5′ genomic regionof the soybean A3244 genome flanking the transgene insert, the insertcomprising a portion of the right border region (RB) from Agrobacteriumtumefaciens, the chimeric promoter FMV/Tsf1 and related linked elements(U.S. Pat. No. 6,660,911; also referred to as FMV/E1F1α) is operablyconnected to an Arabidopsis EPSPS chloroplast transit peptide codingsequence (herein referred to as CTP2 or TS-AtEPSPS CTP2, U.S. Pat. No.5,633,435, operably connected to a glyphosate resistant EPSPS (hereinreferred to as CP4 EPSPS or aroA:CP4, isolated from Agrobacteriumtumefaciens strain CP4 and coding sequence modified for enhancedexpression in plant cells, U.S. Pat. No. 5,633,435), operably connectedto the 3′ termination, region from pea ribulose 1,5-bisphosphatecarboxylase (herein referred to as E9 3′ or T-Ps.RbcS:E9, Coruzzi etal., (1984), a portion of the left border (LB) region from Agrobacteriumtumefaciens, and the 3′ genomic region of the soybean A3244 genomeflanking the transgene insert. DNA molecules useful as primers in DNAamplification methods can be derived from the sequences of the geneticelements of the transgene insert contained in soybean event MON89788.These primer molecules can be used as part of a primer set that alsoincludes a DNA primer molecule derived from the genome of soybeanflanking the transgene insert. Soybean event MON89788 was produced bytransformation of soybean line A3244 (U.S. Pat. No. 5,659,114) by anAgrobacterium mediated method, for example, methods described in U.S.Pat. Nos. 6,384,301 and 7,002,058 (herein incorporated by reference intheir entirety).

The inventors of the present invention have discovered that a soybeanline comprising the MON89788 T-type genomic region (T-type iscombination of a transgene and the associated haplotype region of aplant genome) in its genome has an improved yield relative to a linecomprising the previous 40-3-2 T-type genomic region. This wasdemonstrated in replicated field trials including yield data collectedfrom multiple locations in the United States (U.S. patent applicationSer. No. 60/685,584).

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Production of Amplicon Diagnostic for MON89788Genomic DNA

DNA from transgenic soybean event MON89788 is extracted from tissuecomprising soybean seeds, vegetative tissue, or meal. The DNA isisolated from the tissue using Qiagen's DNeasy Plant Miniprep Kitaccording to the manufacturer's instructions (Qiagen Corp. Valencia,Calif.).

A PCR product is produced that comprises a portion of the genomic DNAflanking the 5′ end of the T-DNA (transfer DNA comprising the transgene)insertion in the genome of a plant comprising MON89788. This DNA productcomprises SEQ ID NO:3. The PCR may be performed using one primerdesigned to hybridize to the genomic DNA sequences flanking the 5′ endof the transgene insert (DNA primer A, SEQ ID NO:5; see FIG. 1) pairedwith a second primer (DNA primer B, SEQ ID NO:6) located in thetransgene promoter region (U.S. Pat. No. 6,660,911, SEQ ID NO:28, hereinincorporated by reference and found within SEQ ID NO:9).

A PCR product is produced from the 3′ end of the transgene insert thatcomprises a portion of the genomic DNA flanking the 3′ end of the T-DNAinsertion in the genome of a plant comprising MON89788 This DNA productcomprises SEQ ID NO:4. PCR may be performed using one primer designed tohybridize to the genomic DNA sequences flanking the 3′ end of the insertof each event (DNA primer D, SEQ ID NO:8) and paired with a secondprimer (DNA primer C, SEQ ID NO:7) located in the T-Ps.RbcS:E9 3′transcription termination sequence at the 3′ end of the insert.

The PCR template includes ˜50 ng of genomic DNA. As a negative control˜50 ng of genomic DNA from the non-transgenic soybean cultivar isutilized. Each PCR reaction contains 5 μl 10× Buffer for REDAccuTaq™ LADNA Polymerase Mix (Sigma-Aldrich, St Louis, Mo.), 200 μM each dNTP(Sigma-Aldrich), 0.4 μM each primer, and 2.5 Units JumpStart™ REDTaq™DNA Polymerase (Sigma-Aldrich) in a 50 μl total volume reaction. The PCRreactions are performed under the following cycling conditions: 1 cycleat 94° C. for 3 minutes (min); 32 or 35 cycles at 94° C. for 30 seconds(s), 58° C. for 30 s, 72° C. for 30 s or 1 min; 1 cycle at 72° C. for 10min.

DNA event primer pairs are used to produce an amplicon diagnostic forMON89788 genomic DNA. These event primer pairs include, but are notlimited to primers A and B (SEQ ID NO:5 and 6) and event primer pairs Cand D (SEQ ID NO: 7 and 8), that are used in the described DNAamplification method. In addition to these primer pairs, any primer pairderived from SEQ ID NO:3 or SEQ ID NO:4, or the complements thereof,that when used in a DNA amplification reaction produces an amplicon thatcomprises SEQ ID NO:1 or SEQ ID NO:2 diagnostic for soybean MON89788event-derived tissue, respectively, may be utilized. DNA amplificationconditions illustrated in Table 1 and Table 2 can be used to produce adiagnostic amplicon for MON89788 using the appropriate event primerpairs. Any modification of these methods used to produce an amplicondiagnostic for MON89788 is within the ordinary skill of the art. Anextract putatively containing DNA of a soybean plant or seed comprisingMON89788, or a product derived from a plant comprising MON89788 thatwhen tested in a DNA amplification method produces an amplicondiagnostic for soybean event MON89788 may be utilized as a template foramplification to determine whether MON89788 is present.

The amplicon is produced by the use of at least one primer sequencederived from SEQ ID NO:3 or SEQ ID NO:4 that when used in a PCR methodproduces a diagnostic amplicon for event MON89788. For example, theproduction of the MON89788 amplicons can be performed using a StratageneRobocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf MastercyclerGradient thermocycler as shown in Table 2, or by methods and apparatusknown to those skilled in the art.

TABLE 1 PCR procedure and reaction mixture conditions for theidentification of soybean MON89788 5′ transgene insert/genomic junctionregion. Step Reagent Amount Comments 1 Nuclease-free water add to finalvolume of 20 μl — 2 10X reaction buffer 2.0 μl 1X final (with MgCl₂)concentration of buffer, 1.5 mM final concentration of MgCl₂ 3 10 mMsolution of dATP, 0.4 μl 200 μM final dCTP, dGTP, and dTTP concentrationof each dNTP 4 Event primer A (SEQ ID NO: 5 0.2 μl 0.1 μM finalresuspended in 1X TE buffer or concentration nuclease-free water to aconcentration of 10 μM) 5 Event primer B (SEQ ID NO: 6 0.2 μl 0.1 μMfinal resuspended in 1X TE buffer or concentration nuclease-free waterto a concentration of 10 μM) 6 RNase, DNase free (500 μg/ml) 0.1 μl 50ng/reaction 7 REDTaq DNA polymerase 1.0 μl (recommended to switch 1unit/reaction (1 unit/μl) pipets prior to next step) 8 Extracted DNA(template): — Samples to be analyzed: individual leaves 10-200 ng ofgenomic DNA pooled leaves 200 ng of genomic DNA (maximum of 10leaves/pool) Negative control 50 ng of non-transgenic soybean genomicDNA Negative control no template DNA (solution in which DNA wasresuspended) Positive control 50 ng of soybean genomic DNA comprisingMON89788

Gently mix and, if needed (no hot top on thermocycler), add 1-2 drops ofmineral oil on top of each reaction. Proceed with the PCR in aStratagene Robocycler (Stratagene, La Jolla, Calif.), MJ Engine(MJR-Biorad, Hercules, Calif.), Perkin-Elmer 9700 (Perkin Elmer, Boston,Mass.), or Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany)thermocycler using the following cycling parameters (Table 2). The MJEngine or Eppendorf Mastercycler Gradient thermocycler should be run inthe calculated mode. Run the Perkin-Elmer 9700 thermocycler with theramp speed set at maximum.

TABLE 2 Thermocycler conditions Cycle No. Settings: StratageneRobocycler 1 94° C. 3 minutes 34 94° C. 1 minute 64° C. 1 minute 72° C.1 minute and 30 seconds 1 72° C. 10 minutes Cycle No. Settings: MJEngine or Perkin-Elmer 9700 1 94° C. 3 minutes 34 94° C. 30 seconds 64°C. 30 seconds 72° C. 1 minute 1 72° C. 10 minutes Cycle No. Settings:Eppendorf Mastercycler Gradient 1 94° C. 3 minutes 34 94° C. 15 seconds64° C. 15 seconds 72° C. 1 minute 1 72° C. 10 minutes

Example 2 Sequence Determination of Transgene/Genomic Region andSouthern Analysis

DNA sequencing of the PCR products provides for DNA that can be used todesign additional DNA molecules as primers and probes for theidentification of soybean plants or seed comprising MON89788. PCRproducts of the expected sizes representing the 5′ and 3′transgene/genomic sequences were isolated by separation of the PCRproducts on a 2.0% agarose gel by electrophoresis. PCR products areisolated that include the 5′ and 3′ DNA regions that span the insertjunction between the transgene insertion into the soybean genome. The 5′and 3′ PCR products for MON89788 are purified by agarose gelelectrophoresis followed by isolation from the agarose matrix using theQIAquick Gel Extraction Kit (catalog #28704, Qiagen Inc., Valencia,Calif.). The purified PCR products are then sequenced (e.g. ABI Prism™377, PE Biosystems, Foster City, Calif.) and analyzed (e.g. DNASTARsequence analysis software, DNASTAR Inc., Madison, Wis.).

A DNA sequence was determined for the nucleotide base pair segmentrepresenting the transgene/genomic region of event MON89788 asillustrated in FIG. 1 and identified as SEQ ID NO:9. The genomic andtransgene elements that are contained in SEQ ID NO:9 are described inTable 3. The 5′ and 3′ flanking regions are included in SEQ ID NO:9 andgiven in SEQ ID NOs:21 and 22.

The junction sequences are relatively short polynucleotide moleculesthat are novel DNA sequences and are diagnostic for MON89788 DNA whendetected in a polynucleic acid detection assay. The junction sequencesin SEQ ID NO:1 and SEQ ID NO:2 represent 10 polynucleotides on each sideof an insertion site of the transgene fragment and soybean genomic DNAin MON89788. Longer or shorter polynucleotide junction sequences can beselected from SEQ ID NO:3 or SEQ ID NO:4. The junction molecules (5′junction region SEQ ID NO:1, and 3′ junction region SEQ ID NO:2) areuseful as DNA probes or as DNA primer molecules in methods for DNAdetection.

Primers and probes used in a Taqman® method (Roche Molecular Systems,Inc., Pleasanton, Calif.) for detection of an event specific DNAmolecule were developed for event MON89788. The primer molecules arereferred to as SQ2824 (SEQ ID NO:10), SQ2826 (SEQ ID NO:11), SQ1141 (SEQID NO:12), SQ1142 (SEQ ID NO:13), SQ5543 (SEQ ID NO:14) and the probemolecules are referred to as PB871-6FAM (SEQ ID NO:15), PB2191-VIC (SEQID NO:16), and PB57-VIC (SEQ ID NO:17). The primers and probes were usedin the Taqman® method according to manufacturers instructions to providea diagnostic amplicon for DNA comprising MON89788. Soybean tissuesincluding processed products, for example meal, are useful sources ofDNA for this method. Additional primers used to produce an amplicon fromsoymeal include SEQ ID NOs:18-20.

TABLE 3 Genome and genetic element annotation of the transgene/genomicDNA fragment (SEQ ID NO: 9) contained in the genome of soybeancomprising MON89788. Location in Genetic Element¹ Sequence² Function(Reference) Sequence flanking 5′   1-1103 SOYBEAN GENOMIC DNA end of theinsert 5′Junction region 1093-1113 DNA region spanning the transgeneinsertion B³-Right Border 1104-1145 DNA region from Agrobacteriumtumefaciens containing the right border sequence used for transfer ofthe T-DNA (Depicker et al., 1982) Intervening Sequence 1146-1215Sequences used in DNA cloning P⁴-FMV/Tsf1 1216-2255 Chimeric promoterconsisting of enhancer sequences from the 35S promoter of the FigwortMosaic virus (Richins et al., 1987) and the promoter from the Tsf1 geneof Arabidopsis thaliana (encoding elongation factor EF-1alpha (Axelos,et al., 1989) L⁵-Tsf1 2256-2301 5′ nontranslated leader (exon 1) fromthe Tsf1 gene of Arabidopsis thaliana encoding elongation factor EF-1alpha (Axelos et al., 1989) I⁶-Tsf1 2302-2923 Intron from the Tsf1 geneof Arabidopsis thaliana encoding elongation factor EF-1 alpha (Axelos etal., 1989) Intervening Sequence 2924-2932 SEQUENCES USED IN DNA CLONINGTS⁷-CTP2 2933-3160 Sequences encoding the chloroplast transit peptidefrom the ShkG gene of Arabidopsis thaliana encoding EPSPS (Klee et al.,1987) CS⁸-cp4 epsps 3161-4528 Codon optimized coding sequence of thearoA gene from the Agrobacterium sp. strain CP4 encoding the CP4 EPSPSprotein (Padgette et al., 1996; Barry et al., 1997) Intervening Sequence4529-4570 Sequences used in DNA cloning T⁹-E9 4571-5213 3′ nontranslatedsequence from the RbcS2 gene of Pisum sativum encoding the Rubisco smallsubunit (Coruzzi et al., 1984) Intervening Sequence 5214-5256 Sequencesused in DNA cloning B-Left Border 5257-5406 DNA region fromAgrobacterium tumefaciens containing the left border sequence used fortransfer of the T-DNA (Barker et al., 1983) 3′ junction region 5396-5416DNA region spanning the transgene insertion Sequence flanking 3′5407-6466 Soybean genomic DNA end of the insertSouthern Blot Analysis

Genomic DNA from a plant comprising MON89788 and control soybean genomicDNA (˜15 μg of each) is digested with various restriction enzymes (140U) in a total volume of 150 μl including 15 μl of the correspondingmanufacturer's buffer (NEB, Beverly, Mass.). Restriction endonucleases,e.g., Bgl11, BamH1, Nco1, Hind111, and Bcl1, are used in the Southernanalysis of MON89788. Endonuclease digests are performed at theappropriate temperature for at least 6 hours. After incubating, the DNAis precipitated with 3M sodium acetate and 2.5 volumes of ethanol.Subsequently, the DNA is washed with 70% ethanol, dried, and resuspendedin 40 μl of TBE. Loading buffer (0.2×) is added to the samples and thensubjected to electrophoresis on agarose gels (0.8%) for 16-18 hours at30 volts. The gels are stained with ethidium-bromide, then treated witha depurination solution (0.125N HCL) for 10 minutes, with a denaturingsolution (0.5M sodium hydroxide, 1.5M sodium chloride) for 30 minutes,and finally with a neutralizing solution (0.5M Trizma base, 1.5M sodiumchloride) for 30 minutes. The DNA is transferred to Hybond-N membrane(Amersham Pharmacia Biotech, Buckingamshire, England) using aTurboblotter (Schleicher and Schuell, Dassel, Germany) for 4-6 hours andthen fixed to the membrane using a UV light.

Membranes are prehybridized with 20 mls of DIG Easy Hyb solution (RocheMolecular Biochemicals, Indianapolis, Ind.; cat. #1603558) for 2-4 hoursat 45° C. Radioactive DNA probes (³²P dCTP) homologous or complementaryto SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or aportion thereof are made using a Radprime DNA Labeling kit (Invitrogen,Carlsbad, Calif.; cat. #18428-011). Unincorporated nucleotides areremoved using SEPHADEX G-50 columns (Invitrogen). The prehybridizationsolution is replaced with 10 mls of pre-warmed DIG Easy Hyb solutioncontaining the denatured probe to a final concentration of 1 millioncounts per ml. The blots are hybridized at 45° C. for 16-18 hours.

Blots are washed with a low stringency solution (5×SSC, 0.1×SDS) at 45°C. and then repeatedly washed with a higher stringency solution(0.1×SSC, 0.1% SDS) at 65° C. The blots are exposed to a phosphor screen(Amersham Biosciences, Piscataway, N.J.) for >2 hours and the exposureread using a Data Storm 860 machine (Amersham Biosciences). Thesemethods and conditions exemplified may be modified by those skilled inthe art of detecting DNA in a sample.

Example 3 Weed Control

Controlling the growth of weeds in a field of soybeans comprisingMON89788. A field is planted with soybean seeds comprising MON89788, theseeds are allowed to germinate into plants and the field of plants istreated with a herbicide formulation containing glyphosate. An effectivedose of a glyphosate formulation at treatment rates of from about 0.25lb ae/A (pounds of glyphosate acid equivalent/acre) to 3 or more lb ae/Ais applied to the field. Rates often applied range from about 0.75 lbae/A to 1.5 lb ae/A at a frequency of one or more treatments during thegrowing season as necessary to control the growth of weeds in a field.Seeds from the plants comprising MON89788 are harvested from the treatedplants.

A deposit of the Monsanto Technology LLC, soybean seed representative ofevent MON89788 disclosed above and recited in the claims has been madeunder the Budapest Treaty with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110. The ATCCaccession number for the deposit comprising event MON89788 (also knownas MON19788 or GM_A19788) is PTA-6708, deposited May 11, 2005. Thedeposit will be maintained in the depository for a period of 30 years,or 5 years after the last request, or for the effective life of thepatent, whichever is longer, and will be replaced as necessary duringthat period.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed:
 1. A transgenic soybean plant, seed, or plant partcomprising a DNA molecule comprising SEQ ID NO: 3 flanking a glyphosatetolerant 5-enolpyruvyl-3-physphoshikimate synthase (EPSPS) codingsequence.
 2. The transgenic soybean plant, seed, or plant part of claim1, further defined as a soybean plant.
 3. The transgenic soybean plant,seed, or plant part of claim 1, further defined as a soybean seed. 4.The transgenic soybean plant, seed, or plant part of claim 1, furtherdefined as a soybean plant part.
 5. A transgenic soybean plant, seed, orplant part comprising a DNA molecule comprising SEQ ID NO: 4 flanking aglyphosate tolerant 5-enolpyruvyl-3-physphoshikimate synthase (EPSPS)coding sequence.
 6. The transgenic soybean plant, seed, or plant part ofclaim 5, further defined as a soybean plant.
 7. The transgenic soybeanplant, seed, or plant part of claim 5, further defined as a soybeanseed.
 8. The transgenic soybean plant, seed, or plant part of claim 5,further defined as a soybean plant part.
 9. A transgenic soybean plant,seed, or plant part comprising a DNA molecule comprising SEQ ID NO: 9.10. The transgenic soybean plant, seed, or plant part of claim 9,further defined as a soybean plant.
 11. The transgenic soybean plant,seed, or plant part of claim 9, further defined as a soybean seed. 12.The transgenic soybean plant, seed, or plant part of claim 9, furtherdefined as a soybean plant part.